Identification of mutations in human smooth muscle actin {ACTA2) and myosin strongly suggest that disruption of smooth muscle cell contractile function predisposes individuals to thoracic aortic aneurysms and aortic dissections (TAAD). Project 1 seeks to determine the molecular mechanisms by which ACTA2 missense mutations lead to TAAD and occlusive vascular diseases. The hypothesis is that ACTA2 mutations lead to altered force output and energetic overload of smooth muscle cells, likely contributing to the eventual death of the cells. This proposal depends on our demonstrated ability to express human smooth muscle actin and myosin in Sf9 cells. In Aim 1 we will assess if ACrA2 missense mutations alter filament assembly or the structural integrity of the filament. Single filament polymerization assays using total internal reflection fluorescence (TIRF) microscopy will be used to follow assembly and disassembly of individual filaments as a function of time, which will identify mutants with polymerization defects. The ability of the mutant actins to copolymerize with WT will be tested. Altered structural integrity of the filament will be evaluated by flexural rigidity measurements. Actin acts as a structural element through which force is transmitted, so even subtle alterations in the filament structure could have profound effects on force production by myosin. In Aim 2 it will be determined if ACTA2 missense mutations affect the ability of myosin to generate motion and force. The interaction of actin with smooth muscle myosin will be assessed by an unloaded ensemble motility assay, which reflects the kinetics of the actomyosin interaction. Mutants will be further characterized under load using a force-clamp laser trap assay, which involves single actin filaments interacting with a small ensemble of myosin at different constant loads. These measurements will provide the average maximum isometric force generated by myosin. Decreased power output could trigger compensatory mechanisms in the smooth muscle cell. Homo and heteropolymers will be compared, as well as pure actin versus actin- tropomyosin. Aim 3 determines if ACTA2 missense mutations alter actin filament structure, or the conformational dynamics of the filament. Actin filament structure will first be analyzed by electron microscopy of negatively stained filaments. High resolution cryo-electron microscopy (collaboration with Dr. Egelman) will then be used to test if the actin mutations interfere with the conformational dynamics of actin. This possibility is likely given that many of the mutations are buried, and cannot directly affect the interaction with myosin or other actin-binding proteins. RELEVANCE (See instructions): Mutations in human smooth muscle actin (ACTA2) and myosin, and in the kinase that activates myosin, have been linked to thoracic aortic aneurysms (TAAD), as well as premature stroke and coronary artery disease. Mutations in ACTA2 are responsible for -15% of reported cases of TAAD. This project seeks to study the effect of the mutations in ACTA2 at the molecular level to allow an understanding of the pathological processes that take place, which in turn could lead to new insights and better treatments for these diseases.